Throughout this application various publications are referenced by full citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Pharmacological studies, and more recently gene cloning, have established that multiple receptor subtypes exist for most, if not all, neurotransmitters. The existence of multiple receptor subtypes provides one mechanism by which a single neurotransmitter can elicit distinct cellular responses. The variation in cellular response can be achieved by the association of individual receptor subtypes with different G proteins and different signalling systems. Further flexibility is provided by the ability of distinct receptors for the same ligand to activate or inhibit the same second messenger system.
Individual receptor subtypes reveal characteristic differences in their abilities to bind a number of ligands, but the structural basis for the distinct ligand-binding properties is not known. Physiologists and pharmacologists have attempted to specify particular biological functions or anatomical locations for some receptor subtypes, but this has met with limited success. Similarly, the biochemical mechanisms by which these receptors transduce signals across the cell surface have been difficult to ascertain without having well-defined cell populations which express exclusively one receptor subtype.
While all the receptors of the serotonin type recognize serotonin, several pharmacologically distinct subtypes of serotonin receptors have been identified, and given a classification name 5-HTX, where X identifies the subtype. In many cases, these subtypes have been or will be associated with single gene products, but in some cases a single subtype may be found to contain several different receptor proteins (gene products) or two different subtypes may be later shown to arise from different properties of the same receptor protein which are exhibited when it is expressed in different tissue environments. In many cases, different serotonin receptor subtypes have been shown to couple to different second messenger pathways that are linked through guanine-nucleotide regulatory (G) proteins.
Radioligand filtration binding techniques have been employed for over ten years in an effort to more completely characterize receptor subtypes within the serotonin receptor family (Schmidt and Peroutka, FASEB J. 3:2242 (1989)). Using these methods, two broad classes of G protein-coupled serotonin receptors have been described, 5-HT1, and 5-HT2. These differ in their selectivity for drugs. 5-HT1 receptors display high (nanomolar) affinity for serotonin and can be labeled with [3H]5-HT. 5-HT2 receptors display low affinity for serotonin but have high (nanomolar) affinity for antagonists such as Ketanserin, Mesulergine, Metergoline and d-LSD.
Within the 5-HT1 receptor class, several subtypes have been distinguished on the basis of their pharmacological binding profiles, second messenger coupling and physiological roles. One such subtype, the 5-HT1D receptor, was originally defined as a particular type of [3H]5-HT binding site in the bovine caudate (Heuring and Peroutka, J. Neurosci. 7:894 (1987)). This definition was not based on properties of a single purified receptor protein or single gene product, but rather was based on experimental observations in a model tissue. As discussed below, later research has shown that there may be multiple receptor proteins (known as subtypes) within this model tissue, all of which contribute to the binding profile that was used to define the 5-HT1D receptor.
The 5-HT1D receptor subtype has been shown to inhibit adenylate cyclase activity (Schoeffter, P. and Hoyer, D., Naunyn-Schmiedeberg""s Arch. Pharmacol. 340:285 (1989)). The 5-HT1D receptor subtype has also been characterized in guinea pig (Waeber, et al. Naunyn-Schmiedeberg""s Arch. Pharmacol. 340:479-485 (1989)), pigeon (Waeber, 1989), pig (Waeber, et al. Naunyn-Schmiedeberg""s Arch. Pharmacol. 377:595-601 (1988)), calf (Waeber, et al. (1988)) and human brain (Waeber, et al. (1988); Herrick-Davis and Titeler, J. Neurochem, 50:1624-1631 (1988)). Among the other serotonin receptor subtypes, the 5-HT1A, and 5-HT1B receptors inhibit adenylate cyclase, and 5-HT1C and 5-HT2 receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides (Schmidt and Peroutka, FASEB J. 3:2242 (1989)).
The pharmacological actions of sumatriptan (GR43175), a new anti-migraine medication under development by Glaxo Pharmaceutical Corp., have been linked to the 5-HT1D receptor site (Peroutka and McCarthy, Eur. J. Pharmacology 163:133 (1989)); Schoeffter and Hoyer, Naunyn-Schmiedeberg Arch. Pharmacology 340:135 (1989)). Recently, one report has shown that the 5-HT1D binding site of piglet caudate could be subdivided into two sites, based on the binding affinities of sumatriptan and 5-carboxamidotryptamine (5-CT) (Sumner and Humphrey, Br. J. Pharmacol. 98:29 (1989)). One of these binding sites, with low affinity for sumatriptan and 5-CT, resembles the 5-HT1E site of human cortex (Leonhardt, Herrick-Davis and Titeler, J. Neurochem. 53:465 (1989)) while the binding site with high affinity for these compounds resembles the classic 5-HT1D receptor, and the site of action of sumatriptan.
Another study, by Xiong and Nelson (Life Sci. 45:1433-1442 (1989)) indicated that a high affinity [3H]5-HT binding site in the rabbit caudate, termed the 5-HT1R binding site, is similar to, but pharmacologically distinct from, the 5-HT1D binding site described in the bovine caudate. These authors presented data indicating that two drugs, spiperone and spirilene, exhibited significantly lower affinity for the 5-HT1R binding site than for the 5-HT1D receptor, and noted several other differences in binding properties between these sites. Investigation of the bovine caudate in light of these findings led to the conclusion that there may be a component of the 5-HT1D receptor in bovine caudate that represents a 5-HT1R binding site. Alternatively, the authors speculated that the 5-HT1D binding site in the bovine caudate may be a heterogenous group of sites with similar properties. As noted by the authors, it is clear that additional work will be need to clarify these issues.
A gene for a G protein-coupled receptor was recently isolated by Libert, et al. from a dog CDNA library (Science 244:569-572, 1989). This gene, termed the RDC4 gene, was not expressed by these authors, and therefore no characterization of the properties of the protein encoded by this gene was made. The dog RDC4 gene was isolated and expressed by the applicants, and was determined by the applicants to encode a 5-HT1D receptor (not published).
The serotonin 5-HT1D receptors belong to a family of receptors which are distinguished by their seven-transmembrane configuration and their functional linkage to G-proteins. This family includes rhodopsin and related opsins (Nathans, J. and Hogness, D. S., Cell 34:807 (1983)), the xcex1 and xcex2 adrenergic receptors (Dohlman, H. G., et al. Biochemistry 26:2657 (1987)), the muscarinic cholinergic receptors (Bonner, T. I., et al., Science 237:527 (1987)), the substance K neuropeptide receptor, (Masu, Y., et al., Nature 329:836 (1987)), the yeast mating factor receptors, (Burkholder, A. C. and Hartwell, L. H., Nucl. Acids Res. 13:8463(1985); Hagan, D. C., et al., Proc. Natl. Acad. Sci. USA 83:1418 (1986)); Nakayama, N. et al., EMBO J. 4:2643 (1985)), and the oncogene c-mas, (Young, et al., Cell 45:711 (1986)). Each of these receptors is thought to transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Dohlman, H. G., et al., Biochemistry 26:2657 (1987); Dohlman, H. G., et al., Biochemistry 27:1813 (1988); O""Dowd, B. F., et al., Ann.Rev. Neurosci., in press).
This invention provides an isolated nucleic acid molecule encoding a human 5-HT1D receptor.
This invention also provides an isolated protein which is a human 5-HT1D receptor.
This invention provides a vector comprising an isolated nucleic acid molecule encoding a human 5-HT1D receptor.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human 5-HT1D receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding a human 5-HT1D receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT1D receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT1D receptor can bind to a human 5-HT1D receptor which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT1D receptor with the ligand under conditions permitting binding of ligands known to bind to the 5-HT1D receptor, detecting the presence of any of the ligand bound to the 5-HT1D receptor, and thereby determining whether the ligand binds to the 5-HT1D receptor.
This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT1D receptor on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT1D receptor on the surface of a cell with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the human 5-HT1D receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT1D receptor.
This invention also provides a method of detecting expression of a 5-HT1D receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT1D receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT1D receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HT1D receptor by the cell.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human 5-HT1D receptor so as to prevent translation of the mRNA molecule.
This invention provides an antibody directed to the human 5-HT1D receptor.
This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT1D receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT1D receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT1D receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT1D receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a 5-HT1D receptor and which hybridizes to mRNA encoding a 5-HT1D receptor thereby reducing its translation.
This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HT1D receptors which comprises producing a transgenic nonhuman animal whose levels of human 5-HT1D receptor expression are varied by use of an inducible promoter which regulates human 5-HT1D receptor expression. This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HT1D receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT1D receptor.
This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT1D receptor allele which comprises: a obtaining DNA of subjects suffering from the disorder; b.performing a restriction digest of the DNA with a panel of restriction enzymes; c.electrophoretically separating the resulting DNA fragments on a sizing gel; d.contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human 5-HT1D receptor and labelled with a detectable marker; e.detecting labelled bands which have hybridized to the the DNA encoding a human 5-HT1D receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f.preparing DNA obtained for diagnosis by steps a-e; and g.comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific human 5-HT1D receptor allele is diagnosed.
This invention provides a method of preparing the isolated 5-HT1D receptor which comprises inducing cells to express 5-HT1D receptor, recovering the receptor from the resulting cells, and purifying the receptor so recovered. This invention provides a method of preparing the isolated 5-HT1D receptor which comprises inserting nucleic acid encoding 5-HT1D receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a receptor so as to prevent translation of the mRNA molecule.
This invention Also provides a transgenic nonhuman mammal expressing DNA encoding a receptor.
This invention further provides a transgenic nonhuman mammal expressing DNA encoding a receptor so mutated as to be incapable of normal receptor activity, and not expressing native receptor.
This invention provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a transgenic nonhuman animal whose levels of the receptor expression are varied by use of an inducible promoter which regulates receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of the receptor.
This invention further provides transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding the receptor and which hybridizes to mRNA encoding the receptor thereby preventing its translation.
This invention provides a method for determining whether a ligand not known to be capable of binding to a receptor can bind to a receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a receptor with the ligand under conditions permitting binding of ligands known to bind to a receptor, detecting the presence of any of the ligand bound to the receptor, and thereby determining whether the ligand binds to a receptor.